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Stanford Report, May 31, 2000

Study of frog tuberculosis may help unlock secrets of human disease  

BY MITCH LESLIE

Frogs get tuberculosis too, and by studying the amphibian version of this disease, Stanford researchers have pinpointed two genes that may enable the TB bacterium to survive for decades within the human body. The findings may help clear up the mystery of how the TB bacterium sets up house within the very immune system cells that are supposed to destroy it.

The bacterium that causes human TB belongs to a notorious family of killers that victimizes many other animals, said Lalita Ramakrishnan, MD, PhD, a senior research scientist in the lab of Stanley Falkow, PhD, professor of microbiology and immunology. One species of bacterium sickens cattle, for example, while another attacks frogs, fishes and other cold-blooded animals.

But like 90 percent of the 2 billion people infected by the human TB bacterium, most infected frogs don't get sick. Instead they develop a lifelong "silent" infection in which the bacteria linger within the body without causing symptoms. However, if the frogs' immune systems are weakened, "they will get rip-roaring disease and die," Ramakrishnan said. The same thing can happen in a person with silent tuberculosis infection whose immune system is then throttled by the AIDS virus, for example.

During this clinically silent or latent period, the bacteria bed down inside the immune system cells known as macrophages, which normally annihilate bacteria and other interlopers. Responding to the invasion, macrophages may huddle with other immune cells to form little nodules called granulomas, or tubercles. Tubercles gave the disease its name and may represent a standoff between the immune system's attempts to wall in the organism and the bacterium's efforts to make a snug home for itself, Ramakrishnan said.

Because of the difficulties of working with human TB, scientists know little about how the bacterium alters its biochemistry to persist within macrophages and tubercles. Ramakrishnan, Falkow and Nancy A. Federspiel, PhD, associate director of the Stanford DNA Sequencing and Technology Center, decided to address the question by finding out which genes were activated in the frog bacterium when it dwells in macrophages or tubercles.

To identify the active genes, the team manipulated the bacteria so that some of their genetic on-off switches were attached to a gene that codes for a glowing green protein. Then they used a machine to pick out the macrophages that contained luminous bacteria. By determining which on-off switches had been turned on, they could identify which genes the bacteria had activated to cope with the hostile environment within a macrophage or tubercle.

They found that when inhabiting a macrophage the bacterium switches on six genes that are not turned on when the bacterium is growing in a laboratory culture. When living in a tubercle, the bacterium switches on an additional four genes. All of the genes are similar to genes in the human TB bacterium -- no surprise, since the two species of bacteria are close relatives. Two of the genes were particularly interesting, Ramakrishnan said, because they belong to "a family of mystery genes" whose function is unknown, even though they constitute about 5 to 10 percent of the bacterium's genetic material.

When the researchers disrupted either of these two genes, they found that the bacteria could no longer reproduce inside macrophages. Disruption of one of the genes also handicapped the bacterium's ability to live within tubercles. "At least some members of this gene family play a role in virulence in this bacterium," Ramakrishnan said.

"It's very likely that the genes we found using this system will be important in human TB as well," she added. She and her colleagues reported their findings in the May 26 issue of Science.

The results demonstrate that frog TB makes a good model system for studying the human disease, Ramakrishnan said. The amphibian bacterium is safer to work with. Because it doesn't travel through the air like the human TB bacterium, no elaborate facilities to prevent infection are required. And the frog bacterium grows five times as fast as its human counterpart.

Although the frog bacterium is not a danger to humans, it can cause mild illness. Reflecting its penchant for cold-blooded hosts, when it invades the human body it seeks the coolest region -- the skin -- producing granulomas there. These infections, which can easily be treated with antibiotics, are common among people who handle fish and among swimmers, Ramakrishnan said.

The work was supported by a grant from the National Institutes of Health. SR